Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection

Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157: H7 for 24+/-1 h at 4 degrees C. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.     

Transfer of Escherichia coli O157:H7 to romaine lettuce due to contact water from melting ice

Ice can be used to chill romaine lettuce and maintain relative humidity during transportation. Escherichia coli O157:H7 may contaminate water used for ice. The objective of this study was to determine the potential for E. coli O157:H7 contamination of romaine lettuce from either ice contaminated with the pathogen or by transfer from lettuce surfaces via melting ice. In experiment 1, lettuce was spot inoculated with E. coli O157:H7 and chilled with ice prepared from uncontaminated tap water. In experiment 2, water inoculated with this pathogen was frozen and used to ice lettuce. Three heads of lettuce were stacked in each container and stored at 4 or 20 degrees C. After the ice melted, E. coli O157:H7 attachment to and recovery from the lettuce leaves were determined. For experiment 1, the population of E. coli O157:H7 attached to inoculated sites averaged 3.8 and 5.5 CFU/cm2 at 4 and 20 degrees C, respectively. Most of the uninoculated sites became contaminated with the pathogen due to ice melt. For experiment 2, 3.5 to 3.8 log CFU E. coli O157:H7 per cm2 was attached to the top leaf on the first head. After rinsing with chlorinated water (200 microg/ml), E. coli O157:H7 remained on the surface of the top head (1.8 to 2.0 log CFU/cm2). There was no difference in numbers of E. coli O157:H7 recovered from each sampling site at 4 and 20 degrees C. Results show that E. coli O157:H7 can be transferred onto other produce layers in shipping containers from melted ice made of contaminated water and from contaminated to uncontaminated leaf surfaces.     

Use of fluorescent microspheres as a tool to investigate bacterial interactions with growing plants

Foodborne pathogens may exist as endophytes of growing plants. The internalization of Escherichia coli O157:H7 or other foodborne pathogens in growing lettuce plants may be independent of microbial factors. Mature lettuce plants were surface irrigated with E. coli O157:H7 or with FluoSpheres (fluorescent microspheres) and harvested 1, 3, and 5 days post-exposure. FluoSpheres were utilized as a bacterial surrogate. Microscopic examination of root, stem, and leaf tissue sections revealed that FluoSpheres were internalized into growing plants. Laser scanning confocal microscopy revealed that FluoSpheres were present within the root tissue and leaf stem tissue. The presence of FluoSpheres in internal portions of stem and leaf tissue suggests transport of the spheres from the root upward into the edible tissue. The level of uptake of FluoSpheres and E. coli O157:H7 was quantified using filtration. Numbers of FluoSpheres and E. coli O157:H7 cells in plant tissue were similar. The entry of E. coli O157:H7 into lettuce plants may be a passive event because the concentration of FluoSpheres was similar to that of the pathogen.     

Penetration of Escherichia coli O157:H7 into lettuce as influenced by modified atmosphere and temperature.

The effects of temperature and atmospheric oxygen concentration on the respiration rate of iceberg lettuce and Escherichia coli O157:H7 cells attachment to and penetration into damaged lettuce tissues were evaluated. Respiration rate of lettuce decreased as the temperature was reduced from 37 to 10 degrees C. Reducing the temperature further to 4 degrees C did not affect the respiration rate of lettuce. Respiration rate was also reduced by lowering the atmospheric oxygen concentration. Lettuce was submerged in E. coli O157:H7 inoculum at 4, 10, 22, or 37 degrees C under 21 or 2.7% oxygen. Attachment and penetration of E. coli O157:H7 were not related to the respiration rate. The greatest numbers of E. coli O157:H7 cells attached to damaged lettuce tissues at 22 degrees C at both oxygen concentrations. More cells were attached under 21% oxygen than under 2.7% oxygen at each temperature, but this difference was small. Penetration of E. coli O157:H7 into lettuce tissue was determined by immunostaining with a fluorescein isothiocyanate-labeled antibody. Under 21% oxygen, E. coli O157:H7 cells showed greatest penetration when lettuce was held at 4 degrees C, compared to 10, 22. or 37 degrees C, and were detected at an average of 101 microm below the surfaces of cut tissues. However, under 2.7% oxygen, there were no differences in degree of penetration among four incubation temperatures. The degree of E. coli O157:H7 penetration into lettuce tissue at 4 or 22 degrees C was greater under 21% oxygen than under 2.7% oxygen; however, no difference was observed at 37 degrees C. Conditions that promote pathogen penetration into tissue could decrease the effectiveness of decontamination treatments.     

Comparison of methods for fluorescent detection of viable, dead, and total Escherichia coli O157:H7 cells in suspensions and on apples using confocal scanning laser microscopy following treatment with sanitizers

The influence of treating Escherichia coli O157:H7 cells labeled with an enhanced green fluorescent protein (EGFP) plasmid with 20 microg/ml active chlorine, 100 mg/ml hydrogen peroxide, and 80 mg/ml acetic acid on fluorescence intensity was determined. In addition, fluorescent staining methods to differentiate viable and dead E. coli O157:H7 cells on the cuticle of Red Delicious cv. apples following treatment with water or 200 microg/ml active chlorine were evaluated. Suspensions of E. coli O157:H7 EGFP+ cells were exposed to chemical treatment solutions for 0, 30, 60, 120, or 300 s before populations (log10 cfu/ml) were determined by surface plating, and fluorescence intensities of suspensions and individual cells were measured using spectrofluorometry and confocal scanning laser microscopy (CSLM), respectively. The relative fluorescence intensity of suspensions and individual cells changed upon exposure to various treatments. Results indicate that the use of EGFP to tag E. coli O157:H7 may not be appropriate for investigations seeking to microscopically differentiate viable and dead cells on produce following surface treatment with sanitizers. SYTOX Orange and SYTOX Green nucleic acid stains fluorescently labeled dead E. coli O157:H7 cells attached to apple cuticles more intensely than did propidium iodide. A cross-signal occurred between CSLM photomultipliers when examining tissues treated with SYTOX Orange to detect dead cells and antibody labeled with Alexa Fluor 488 to detect total (dead and viable) cells. Because of the possibility of cross-signal resulting in an overestimation of the number of dead cells on apples and, perhaps, other produce treated with these stains, SYTOX Green is preferred to detect dead cells and antibody labeled with Alexa Fluor 594 is preferred to detect the total number of cells on apple surfaces following treatment with sanitizers. The performance of SYTOX Green in combination with Alexa Fluor 594 to detect dead and total cells of E. coli O157:H7 on other produce remains to be determined.     

Irradiation compared with chlorination for elimination of Escherichia coli O157:H7 internalized in lettuce leaves: influence of lettuce variety

ABSTRACT:  Pathogenic bacteria internalized in leaf tissues are not effectively removed by surface treatments. Irradiation has been shown to inactivate leaf-internalized bacteria, but many aspects of targeting these protected pathogens remain unknown. Bacterial cells of a cocktail mixture of 3 isolates of Escherichia coli O157:H7 were drawn into the leaves of iceberg, Boston, green leaf, and red leaf lettuce using vacuum perfusion. The inoculated leaves were treated with a 3-min wash with sodium hypochlorite solution (0, 300, or 600 ppm) or various doses of ionizing radiation (0.25 to 1.5 kGy). Leaves were stomached to recover the internalized cells and survivors enumerated. Washes with 0 ppm (water), 300 ppm, and 600 ppm chlorine solutions each gave reductions of less than 1 log. These reductions were statistically significant only in the case of green leaf lettuce. In contrast, irradiation effectively reduced E. coli O157:H7 on all varieties examined, with all doses tested being significantly reduced from the untreated control. The specific variety influenced the efficacy of irradiation. The greatest reduction obtained was 5 logs on iceberg lettuce treated with 1.5 kGy. The D ₁₀ values (the dose necessary to achieve a 1 log reduction) were significantly ( P < 0.05) different among the varieties of lettuce tested, and ranged from 0.30 kGy (iceberg) to 0.45 kGy (Boston). These values were observed to be notably higher than previous irradiation D ₁₀ values for E. coli O157:H7 surface inoculated onto these 4 lettuce varieties. This study has shown that irradiation is able to effectively reduce viable E. coli O157:H7 cells internalized in lettuce, and that the variety of lettuce influences the specific response.     

Suspending lettuce type influences recoverability and radiation sensitivity of Escherichia coli O157:H7

An outbreak strain of Escherichia coli O157:H7 was inoculated onto closely related but structurally distinct types of lettuce (Lactuca sativa): Boston (butterhead lettuce), iceberg (crisphead lettuce), and green leaf and red leaf (colored variants of looseleaf lettuce). The E. coli O157:H7 was inoculated either onto the surface of cut leaf pieces or into a homogenized leaf suspension. Samples were gamma irradiated, and the radiation sensitivity of the inoculated bacteria was expressed as a D-value (the amount of ionizing radiation necessary to reduce the bacterial population by 90% [kGy]). The recovery of bacteria from nonirradiated leaf pieces was also measured. When inoculated onto the leaf surface, E. coli O157:H7 had significantly stronger radiation sensitivity on red leaf lettuce (D = 0.119 +/- 0.004 [standard error]) and green leaf lettuce (D = 0.123 +/- 0.003) than on iceberg lettuce (D = 0.136 +/- 0.004) or Boston lettuce (D = 0.140 +/- 0.003). When E. coli O157:H7 was inoculated into a homogenized leaf suspension, its sensitivity was significantly stronger on iceberg lettuce (D = 0.092 +/- 0.002) than on green leaf lettuce (D = 0.326 +/- 0.012), Boston lettuce (D = 0.331 +/- 0.009), or red leaf lettuce (D = 0.339 +/- 0.010), with a threefold difference. Significantly fewer bacteria were recovered from the surface of iceberg lettuce than from the surfaces of the other types of lettuce examined. Following radiation doses of up to 0.5 kGy, the texture (maximum shear strength) of lettuce leaves was measured along the midrib and along the leaf edge for each type of lettuce. There was no meaningful change in texture for any type of lettuce for either leaf section examined at any dose up to 0.5 kGy. These data show (i) that relatively subtle differences between lettuce types can significantly influence the radiation sensitivity of associated pathogenic bacteria and (ii) that doses of up to 0.5 kGy do not soften lettuce leaves.     

Inhibition of pathogens on fresh produce by ultraviolet energy

Ultraviolet energy at a wavelength of 253.7 nm (UVC) was investigated for its bactericidal effects on the surface of Red Delicious apples, leaf lettuce and tomatoes inoculated with cultures of Salmonella spp. or Escherichia coli O157:H7. Inoculated samples were subjected to different doses ranging from 1.5 to 24 mW/cm(2) of UVC and enumerated on tryptic soy agar plus 0.05 g/l nalidixic acid to determine effective log reductions of microbial populations. UVC applied to apples inoculated with E. coli O157:H7 resulted in the highest log reduction of approximately 3.3 logs at 24 mW/cm(2). Lower log reductions were seen on tomatoes inoculated with Salmonella spp. (2.19 logs) and green leaf lettuce inoculated with both Salmonella spp. and E. coli O157:H7 (2.65 and 2.79, respectively). No significant statistical difference (p>0.05) was seen in the ability of UVC to inactivate a higher population of either Salmonella spp. or E. coli O157:H7 on the surface of green leaf lettuce. No significant difference was seen among the use of different doses applied to the surface of fresh produce for reduction of E. coli O157:H7 or Salmonella spp. (p>0.05). The use of UVC may prove to be beneficial in protecting the safety of fruits and vegetables in conjunction with Good Agricultural Practices and Good Manufacturing Practices.     

Bactericidal activity of isothiocyanate against pathogens on fresh produce

The bactericidal activity of allyl and methyl isothiocyanate (AITC and MITC) was tested with a rifampicin-resistant strain of Salmonella Montevideo and streptomycin-resistant strains of Escherichia coil O157:H7 and Listeria monocytogenes Scott A. Iceberg lettuce inoculated with high (10(7) to 10(8) CFU/g) and low (10(3) to 10(4) CFU/g) concentrations of bacterial pathogens was treated with AITC and MITC in sealed containers at 4 degrees C for 4 days. AITC showed stronger bactericidal activity than MITC against E. coli O157:H7 and Salmonella Montevideo, whereas MITC showed stronger activity against L. monocytogenes than E. coli O157:H7 and Salmonella Montevideo. Up to 8-log reduction occurred with E. coli O157:H7 and Salmonella Montevideo on lettuce following treatment with vapor generated from 400 microl of AITC for 2 and 4 days, respectively. AITC was used to treat tomatoes inoculated with Salmonella Montevideo on stem scars and skin and apples inoculated with E. coli O157:H7 on stem scars. The bactericidal effect of AITC varied with bacteria species and exposure time. Salmonella Montevideo inoculated on tomato skin was more sensitive to AITC than that on stem scars. Treatment with vapor generated from 500 microl of AITC caused an 8-log reduction in bacteria on tomato skin but only a 5-log reduction on tomato stem scars. The bactericidal activity of AITC was weaker for E. coli O157:H7 on apple stem scars; only a 3-log reduction in bacteria occurred when 600 microl of AITC was used.     

Survival of enterohemorrhagic Escherichia coli O157:H7 in bovine feces applied to lettuce and the effectiveness of chlorinated water as a disinfectant.

Bovine feces are a potential vehicle for transmitting enterohemorrhagic Escherichia coli O157:H7 to humans. A study was undertaken to determine survival characteristics of E. coli O157:H7 on iceberg lettuce using 0.1% peptone water and bovine feces as carriers for inocula. Four levels of inoculum, ranging from 10(0) to 10(5) CFU of E. coli O157:H7 per g of lettuce, were applied. Populations surviving on lettuce stored at 4 degrees C were monitored for up to 15 days. Regardless of the type of carrier, viable cells of E. coli O157:H7 were detected on lettuce after 15 days, even when the initial inoculum was 10(0) to 10(1) CFU/g. Spray treatments of lettuce with 200 ppm chlorine solution or deionized water were equally effective in killing or removing E. coli O157:H7 from lettuce. Holding lettuce for 5 min after spray treatment was not more effective in reducing populations than holding for 1 min before rinsing with water. Prevention of contamination of lettuce with bovine feces that may harbor E. coli O157:H7 as well as other infectious microorganisms is essential to minimizing the risk of illness. The development of sanitizers more efficacious than chlorine for the removal of pathogens from raw fruits and vegetable is needed.     

Direct microscopic observation of lettuce leaf decontamination with a prototype fruit and vegetable washing solution and 1% NaCl-NaHCO3

Efficacy of a prototype, food-grade alkaline surfactant washing solution and 1% NaCl-NaHCO3 (pH 10.0) against Escherichia coli O157:H7 cells on lettuce leaves was evaluated. Lettuce was inoculated with 10(9) CFU/ml of E. coli O157:H7 for 24 +/- 1 h at 4 degrees C. Samples were rinsed and treated with the prototype washing solution containing lauryl sodium sulfate or NaCl-NaHCO3 for 3 min at 22 degrees C. Viability of E. coli O157:H7 cells was examined by plate counts at the surface and cut edge, and by confocal scanning microscopic (CSLM) observation of samples stained with Sytox green and Alexa 594 conjugated antibody against E. coli O157:H7 at intact leaf surface, stomata, and damaged tissue (0 to 10, 30 to 40, and 0 to 40 microm from the cut surface). Although both treatments caused significant log reductions in CFU at the surface and cut edge, log reductions were greater for the prototype washing solution (0.7 to 1.1 log CFU/cm2) than for NaCl-NaHCO3 (0.2 to 0.4 log CFU/cm2) (P < 0.05). Percentage of viability determined by CSLM for prototype washing solution was significantly greater at 30 to 40 microm from cut surfaces than at 0 to 10 and 0 to 40 microm from cut surfaces and intact surfaces (P < 0.05). Stomata provided moderate protection. NaCl-NaHCO3 was less effective than the prototype washing solution, and high percentages of E. coli O157:H7 cells remained viable at all sites except at the surface. The percent viabilities determined by CSLM were not significantly different from those determined by plate counts for NaCl-NaHCO3 treatment (P > 0.05). However, CSLM indicated significantly greater percent viability than plate counts for lettuce treated with the prototype washing solution (P < 0.05). Surfactant-containing washing solutions warrant additional testing for decontamination of fresh produce.     

Effects of environmental stresses on the activities of the uspA, grpE and rpoS promoters of Escherichia coli O157:H7

Heat shock proteins and RNA polymerase sigma factor play an important role in protecting cells against environmental stresses, including starvation, osmotic and oxidative stresses, and cold shock. In this study, the effect of environmental stresses on activity of the auto-fluorescent Escherichia coli O157:H7 generated by the fusion of gfp(uv) to E. coli uspA, grpE and rpoS promoters were examined. Osmotic shock caused about a 4-fold increase in green fluorescence of E. coli O157:H7 harboring uspA::gfp(uv) or rpoS::gfp(uv) at 37 degrees C and room temperature whereas osmotic shock at 5 degrees C did not induce green fluorescence. When starved, E. coli O157:H7 possessing grpE::gfp(uv) was more sensitive for evaluating stress at low temperature while uspA::gfp(uv) was better suited for detecting the stress response at higher temperature. The uspA, grpE and rpoS promoters were up-regulated to varying degrees by stresses commonly encountered during food processing.     

Influence of inoculation method, spot inoculation site, and inoculation size on the efficacy of acidic electrolyzed water against pathogens on lettuce

The influence of bacterial inoculation methods on the efficacy of sanitizers against pathogens was examined. Dip and spot inoculation methods were employed in this study to evaluate the effectiveness of acidic electrolyzed water (AcEW) and chlorinated water (200 ppm free available chlorine) against Escherichia coli O157:H7 and Salmonella spp. Ten pieces of lettuce leaf (5 by 5 cm) were inoculated by each method then immersed in 1.5 liters of AcEW, chlorinated water, or sterile distilled water for 1 min with agitation (150 rpm) at room temperature. The outer (abaxial) and inner (adaxial) surfaces of the lettuce leaf were distinguished in the spot inoculation. Initial inoculated pathogen population was in the range 7.3 to 7.8 log CFU/g. Treatment with AcEW and chlorinated water resulted in a 1 log CFU/g or less reduction of E. coli O157:H7 and Salmonella populations inoculated with the dip method. Spot inoculation of the inner surface of the lettuce leaf with AcEW and chlorinated water reduced the number of E. coli O157:H7 and Salmonella by approximately 2.7 and 2.5 log CFU/g, respectively. Spot inoculation of the outer surface of the lettuce leaf with both sanitizers resulted in approximately 4.6 and 4.4 log CFU/g reductions of E. coli O157:H7 and Salmonella, respectively. The influence of inoculation population size was also examined. Each sanitizer could not completely eliminate the pathogens when E. coli O157:H7 and Salmonella cells inoculated on the lettuce were of low population size (10(3) to 10(4) CFU/g), regardless of the inoculation technique.     

Determination of free chlorine concentrations needed to prevent Escherichia coli O157:H7 cross-contamination during fresh-cut produce wash

This study was conducted to investigate the effect of free chlorine concentrations in wash water on Escherichia coli O157:H7 reduction, survival, and transference during washing of fresh-cut lettuce. The effectiveness of rewashing for inactivation of E. coli O157:H7 on newly cross-contaminated produce previously washed with solutions containing an insufficient amount of chlorine also was assessed. Results indicate that solutions containing a minimum of 0.5 mg/liter free chlorine were effective for inactivating E. coli O157:H7 in suspension to below the detection level. However, the presence of 1 mg/liter free chlorine in the wash solution before washing was insufficient to prevent E. coli O157:H7 survival and transfer during washing because the introduction of cut lettuce to the wash system quickly depleted the free chlorine. Although no E. coli O157:H7 was detected in the wash solution containing 5 mg/liter free chlorine before washing a mix of inoculated and uninoculated lettuce, low numbers of E. coli O157:H7 cells were detected on uninoculated lettuce in four of the seven experimental trials. When the prewash free chlorine concentration was increased to 10 mg/liter or greater, no E. coli O157:H7 transfer was detected. Furthermore, although rewashing newly cross-contaminated lettuce in 50 mg/liter free chlorine for 30 s significantly reduced (P = 0.002) the E. coli O157:H7 populations, it failed to eliminate E. coli O157:H7 on lettuce. This finding suggests that rewashing is not an effective way to correct for process failure, and maintaining a sufficient free chlorine concentration in the wash solution is critical for preventing pathogen cross-contamination.     

Differentiation of viable and dead Escherichia coli O157:H7 cells on and in apple structures and tissues following chlorine treatment

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25 degrees C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4 degrees C, followed by treatment in water or chlorine solution at 21 degrees C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers. as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodgedin protective structures on the surface and within tissues of apples.     

Efficacy of acidic electrolyzed water ice for pathogen control on lettuce

Acidic electrolyzed water (AcEW) was used as frozen AcEW (AcEW-ice) for inactivation of Listeria monocytogenes and Escherichia coli O157:H7 on lettuce. AcEW-ice was prepared from AcEW with 20, 50, 100, and 200 ppm of available chlorine by freezing at -40 degrees C and generated 30, 70, 150, and 240 ppm of chlorine gas (Cl2), respectively. The AcEW-ice was placed into styrene-foam containers with lettuce samples at 20 degrees C for 24 h. Although AcEW-ice generating 30 ppm Cl2 had no effect on L. monocytogenes cell counts, AcEW-ice generating 70 to 240 ppm of Cl2 significantly (P < 0.05) reduced L. monocytogenes by ca. 1.5 log CFU/g. E. coli O157:H7 cell counts were reduced by 1.0 log CFU/g with AcEW-ice generating 30 ppm of Cl2. AcEW-ice generating 70 and 150 ppm of Cl2 reduced E. coli O157:H7 by 2.0 log CFU/g. Further significant reduction of E. coli O157:H7 (2.5 log CFU/g) was demonstrated by treatment with AcEW-ice generating 240 ppm of Cl2. However, treatment with AcEW-ice generating 240 ppm of Cl2 resulted in a physiological disorder resembling leaf burn. AcEW-ice that generated less than 150 ppm of Cl2 had no effect on the surface color of the lettuce. AcEW-ice, regardless of the concentration of the emission of Cl2, had no effect on the ascorbic acid content in the lettuce. The weight ratio of lettuce to AcEW-ice required was determined to be over 1:10. The bactericidal effect of AcEW-ice appeared within the first 2 h. The use of AcEW-ice provides simultaneously for low temperature storage and inactivation of bacteria.     

Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Pseudomonas fluorescens to lettuce leaves

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.     

Recirculating immunomagnetic separation and optimal enrichment conditions for enhanced detection and recovery of low levels of Escherichia coli O157:H7 from fresh leafy produce and surface water

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (C(T) values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42 degrees C for 5 h provided better detection than enrichment at 37 degrees C. Extended incubation of surface water for 20 h at 42 degrees C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.     

Difference between Escherichia coli O157:H7 and non-pathogenic E. coli: survival and growth in seasonings

We examined the survival and growth of Escherichia coli O157:H7 cells incubated with several seasonings, in comparison with those of non-pathogenic E. coli. The cells were incubated at 25 degrees C for 24 h with several concentrations of NaCl, sucrose, soy sauce, worcester sauce and tomato ketchup, and their survival ratios were determined. The E. coli O157:H7 strains showed relatively higher survival ratios in 0.5-1.0 M sucrose, 25% soy sauce and 12.5-50% worcester sauce than the non-pathogenic strains, but slightly lower survival ratios in 0.5-2.0 M NaCl. A noteworthy difference between E. coli O157:H7 and the non-pathogenic strains was that incubation in the presence of 12.5% soy sauce allowed the growth of E. coli O157:H7 strains but reduced the viable cell numbers of non-pathogenic E. coli strains.     

大气压冷等离子体处理对果蔬表面E.coli O157:H7生物膜的清除作用

本文探究了大气压冷等离子体(ACP)处理对E. coli O157:H7生物膜清除作用的最佳处理功率和处理时间,并进一步探究了ACP处理对E. coli O157:H7生物膜的抗菌机制,利用场发射扫描电镜观察了ACP处理前后生物膜形态的变化,最后将ACP处理应用到四种果蔬表面E. coli O157:H7生物膜的清除上。结果表明,在最佳处理功率400 W,最佳处理时间3 min下,ACP通过抑制胞外聚合物中多糖及蛋白质的合成与分泌来抑制生物膜的形成,并在处理当天分别对户太葡萄、圣女果、维多利亚青提及生菜这四种果蔬表面清除98.99%±0.38%、99.92%±0.20%、96.84%±0.18%、99.80%±0.23%的E. coli O157:H7生物膜,在5 d内仍表现出了较好的抑制效果,延长了四种果蔬的贮藏期。结合感官评定结果,ACP处理虽对四种果蔬的色泽和感官品质略有影响,但仍能被大家所接受。综上,ACP处理可在基本不影响四种果蔬的色泽及感官品质前提下,对果蔬表面的E. coli O157:H7生物膜有明显的清除效果。